Phillip H. Klesius

Genetic fingerprinting of Flavobacterium columnare isolates from cultured fish

Aims:  To evaluate the intraspecific diversity of the fish pathogen Flavobacterium columnare

Efficacy of single and combined Streptococcus iniae isolate vaccine administered by intraperitoneal and intramuscular routes in tilapia /Oreochromis niloticus

We evaluated the effectiveness of Streptococcus iniae vaccines prepared from formalin-killed .

Effect of soybean meal replacement by cottonseed meal and iron supplementation on growth, immune response and resistance of Channel Catfish (Ictalurus puctatus) to Edwardsiella ictaluri challenge

Abstract Three basal diets containing 0%, 27.5% and 55.0% solvent-extracted cottonseed meal (CSM) as replacements of 0%, 50% and 100% of solvent-extracted soybean meal (SBM) on an equal nitrogen basis were each supplemented with three levels of iron (40, 336 and 671 mg/kg) from ferrous sulfate heptahydrate (3×3 factorial experiment). Each diet was fed to juvenile channel catfish in triplicate aquaria twice daily to apparent satiation for 10 weeks for subsequent determination of growth response, hematology, specific and non-specific immune response, and mortality following Edwardsiella ictaluri challenge. Fish fed diets containing 27.5% CSM as a replacement of 50% of SBM had improved weight gain (WG) and feed efficiency ratio (FER). Total replacement of SBM by 55.0% CSM decreased WG, feed intake (FI) and FER. Total cell count (TCC), red blood cell count (RBC), hematocrit (Ht) and hemoglobin (Hb) were not affected by dietary levels of CSM. Iron supplementation significantly affected TCC and RBC and maximum values of these parameters were obtained at 336 mg of iron/kg diet. However, Ht and Hb were not affected by increasing levels of supplemental iron. Values for TCC, RBC and Hb were significantly affected by the interaction between dietary levels of CSM and iron. For fish fed the diet containing 0% CSM (SBM-based diets), these parameters increased linearly with increasing dietary levels of iron. When CSM levels were increased to 27.5% or higher, 336 mg supplemental iron was sufficient for maximum hematological values. Macrophage chemotaxis in the presence of exoantigen was significantly higher for fish fed diets containing 55.0% CSM as compared to those fed the lower CSM diets. Agglutinating antibody titers were also significantly higher for fish fed diets containing CSM, but the values did not differ for those fed the 27.5% or 55.0% CSM diets. Dietary levels of iron, and interactions between dietary levels of iron and CSM had no effect on macrophage chemotaxis and antibody titers. Cumulative mortality at 15 days post-challenge was significantly higher for fish fed the SBM-based diet (0% CSM) at 54.4% as compared to 35.0% and 21.6% for those fed the 27.5% and 55.0% CSM diets, respectively. No differences were observed among mortality of fish fed the CSM-containing diets. Dietary levels of iron supplementation, and the interactions between dietary levels of iron and CSM had no effect on post-challenged mortality of fish.

Survival and Antibody Response of Channel Catfish, Blue Catfish, and Channel Catfish Female × Blue Catfish Male Hybrids after Exposure to Edwardsiella ictaluri

Abstract Juvenile Norris strain channel catfish Ictalurus punctatus, blue catfish I. furcatus, and Norris strain channel catfish female × blue catfish male hybrids were challenged with Edwardsiella ictaluri by bath immersion or intraperitoneal injection (high or low dose) in aquaria. Survival (%) after bath immersion was highest for blue catfish (89.5 ± 2.8), intermediate for hybrids (73.8 ± 6.7), and lowest for channel catfish (62.0 ± 4.2). Prechallenge antibody levels to E. ictaluri, measured by enzyme-linked immunosorbent assay, were negative (mean ± SE optical density [OD] = 0.010 ± 0.003). Postchallenge antibody response for blue catfish (OD = 0.132 ± 0.045) was significantly lower than that of channel catfish (OD = 0.350 ± 0.045), whereas the response of the channel × blue catfish F1 hybrids (OD = 0.263 ± 0.051) was intermediate and not significantly different from either parental species. Intraperitoneal injections of E. ictaluri resulted in significant mortality only in channel catfish (88.3 ± 2.6...

Density and dose: factors affecting mortality of Streptococcus iniae infected tilapia /Oreochromis niloticus

Fish density and infectious dose have been suspected to affect the mortality rate of cultured fish exposed to Streptococcusiniae. We determined the effects of S. iniae dose and tilapia (Oreochromisniloticus) density on streptococcal disease mortality. Tilapia with a mean weight of 12.7 g were used and maintained at 25±1°C in aquaria supplied with flow-through water at 0.5 l/min with a 12 h light:12 h dark cycle. Density and dose were evaluated by stocking tilapia at low (5.6 g/l), medium (11.2 g/l) and high (22.4 g/l) density and administering 2.5×107, 5×107 and 1×108 colony-forming units (CFU)/ml of S. iniae by immersion (5 tanks per density and dose, 45 total tanks). Mortality was monitored for 28 days post challenge. A significant difference (P<0.05) was seen in mortality when comparing low (4.8%) and medium (28.4%) and low and high (25.6%) density treatments. No significant difference was observed when comparing medium- and high-density treatments. Two-way analysis of variance demonstrated density had a significant effect on S. iniae mortality (P=0.0001). Doses had little effect on mortality, except at high density by dose which did show a significant interaction (P=0.001). We have demonstrated density has a significant effect on streptococcal disease mortality in tilapia exposed to S. iniae by immersion. We also evaluated infection of susceptible tilapia using dead/moribund S. iniae infected fish (i.e., cohabitation by placing five dead/moribund fish into tanks for 48 h). No significant difference in mortality pattern was observed between immersion in 8.6×107 CFU/ml S. iniae (37.6% and 34.6%) and cohabitation with S. iniae infected tilapia (24.0%). Although, densities used were less than in most water-reuse production systems (30–290 g/l), tilapia density of 11.2 g/l and above was an important factor in mortality of tilapia infected with S. iniae. A health-management strategy would be to reduce fish density thus lowering streptococcal disease mortality.

Nutritional value of heat-treated soybean meal for channel catfish (Ictalurus punctatus)

A study was undertaken to evaluate the effect of heat treatment of defatted raw soybean meal (RSBM) on the growth performance, hematology, immune response and resistance of channel catfish to Edwardsiella ictaluri challenge. Six practical-type diets were formulated to be isocaloric (2.8 kcal DE/kg diet) and isonitrogenous (34% crude protein). A diet containing 45% of commercial soybean meal (CSBM) served as the control. RSBM, non-heat treated (RSBM0) and heated in an autoclave using the dry cycle at 130 °C and 22 psi for 5 (RSBM5), 10 (RSBM10), 20 (RSBM20), and 40 min (RSBM40), was used to isonitrogenously replace the CSBM in the control diet. Each diet was fed to juvenile catfish (4.98 g) in triplicate aquaria twice daily to apparent satiation for 10 weeks. Another batch of diets containing 1% of chromic oxide was used for measurement of apparent digestibility coefficients. Heating RSBM for 20 min or longer lowered trypsin inhibitor (TI) content and increased the apparent protein digestibility (APD). The protein dispersibility index (PDI) decreased with an increase in the duration of heat treatment. Fish fed CSBM and RSBM40 diets had similar weight gain, protein efficiency ratio and apparent protein utilization which were significantly higher than those of fish fed the other diets. Feed intake significantly increased when RSBM was heated for 20 min or longer. Fish fed the RSBM40 diet had significantly lower feed intake but higher feed efficiency than those fed the CSBM diet. Whole-body protein was highest for fish fed the RSBM40 diet but did not differ from that of fish fed the CSBM diet. Hepatosomatic and visceral indices (HSI and VI) of the groups fed CSBM and RSBM40 diets were significantly lower than those fed other diets. Total cell count, red blood cell count, hematocrit and hemoglobin were not significantly affected by dietary treatment. Plasma lysozyme activity and protein of fish fed the CSBM and RSBM40 diets were similar and significantly higher than those of fish fed the other diets. Cumulative mortality 14-day post challenge with E. ictaluri was significantly lower for fish fed the RSMB5 and RSBM10 diets but significantly increased when heating time was increased to 40 min. Macrophage chemotaxis in the presence of exoantigen and antibody titer against E. ictaluri was higher for the groups fed the RSBM containing diets than those fed the CSBM diet, although the differences were not always significant. Results of this study indicate that autoclaving the RSBM for 40 min lowered the TI and PDI, improved the nutritional value of RSBM and increased plasma lysozyme and protein. However, this level of heat treatment significantly decreased macrophage chemotaxis, antibody titer and resistance of fish to E. ictaluri challenge.

Effect of size and temperature on the quantity of immunoglobulin in channel catfish, Ictalurus punctatus

Concentrations of serum immunoglobulin (Ig) were determined for 30 channel catfish from pond water at 10 degrees C. These values were compared to measurements of 15 channel catfish from pond water at 30 degrees C. Channel catfish from 10 degrees C pond water had no significant (P greater than 0.05) different Ig concentrations (mean, 398 mg/dl) than catfish from 30 degrees C pond water (mean, 367 mg/dl). Serum Ig concentrations appear not to be different in cold (10 degrees C) vs warm (30 degrees C) pond water for 37.5-45 cm catfish. Channel catfish, 7.5-15 cm (n = 24) had significantly (P less than 0.05) lower Ig levels (mean, 104 mg/dl) than catfish either 7.5-25.5 cm (n = 57, mean, 232 mg/dl) or 37.5-45 cm (n = 45, mean, 388 mg/dl). Also, catfish 17.5-25.5 cm had a significantly (P less than 0.05) less Ig than catfish 37.5-45 cm. The concentrations of serum Ig increase with size (P = 0.0001) of catfish. The mean Ig concentration for 7.5-45 cm catfish (n = 126) was 263 mg/dl. The Ig concentration range was 44 to 650 mg/dl of serum.

Phylogenetic relationships among Streptococcus agalactiae isolated from piscine, dolphin, bovine and human sources: a dolphin and piscine lineage associated with a fish epidemic in Kuwait is also associated with human neonatal infections in Japan

Streptococcus agalactiae, commonly known as group B streptococcus (GBS), is a cause of infectious disease in numerous animal species. This study examined the genetic relatedness of piscine, dolphin and human GBS isolates and bovine GBS reference strains from different geographical regions using serological and molecular serotyping and multilocus sequence typing (MLST) techniques. Piscine isolates originating from Kuwait, Brazil, Israel and the USA were capsular serotype Ia, a serotype previously unreported in GBS isolated from fish. Sequence typing of piscine isolates produced six sequence types (ST-7, ST-257, ST-258, ST-259, ST-260 and ST-261), the latter five representing allelic designations and allelic combinations not previously reported in the S. agalactiae MLST database. Genomic diversity existed between dolphin and piscine GBS isolates from Kuwait and other geographical areas. Piscine GBS isolates from Brazil, Israel, Honduras and the USA appeared to represent a distinct genetic population of strains that were largely unrelated to human and bovine GBS. The Kuwait dolphin and piscine lineage (ST-7, Ia) was also associated with human neonatal infections in Japan. Comparative genomics of piscine, human and bovine GBS could help clarify those genes important for host tropism, the emergence of unique pathogenic clones and whether these hosts act as reservoirs of one anothers pathogenic lineages.

Growth performance and immune response of channel catfish (Ictalurus puctatus) fed diets containing graded levels of gossypol–acetic acid

Abstract This study was conducted to evaluate the effect of gossypol levels on growth performance, body composition, hematology, immune response and resistance of channel catfish to Edwadsiella ictaluri challenge. A purified basal diet supplemented with 0, 300, 600, 900, 1200 and 1500 mg of gossypol from gossypol–acetic acid was fed to juvenile channel catfish in quadruplicate aquaria to apparent satiation twice daily for 12 weeks. Final weight gain was inversely related to the concentration of dietary gossypol. Fish fed diets without and with 1500-mg gossypol/kg had significantly highest and lowest weight gain, respectively. Feed intake and feed efficiency were a reflection of weight gain. Survival was not affected by dietary levels of gossypol. Whole body moisture increased whereas lipid decreased with increasing dietary gossypol concentrations. Body protein significantly decreased in fish fed with 1500-mg gossypol diet, but did not differ among fish fed with other diets. Body ash did not differ in fish fed diets containing 0–900-mg gossypol/kg but was significantly higher in fish fed higher levels of dietary gossypol. Gossypol concentration in liver was linearly related ( R 2 =0.991) to dietary levels of gossypol. Ratio of (+) to (−) gossypol isomers in liver of fish fed with different diets was relatively constant. Red blood cell (RBC) count of fish fed with the two highest levels of dietary gossypol (1200 and 1500 mg) was significantly lower than those of fish fed with control diet. Hemoglobin significantly decreased in fish fed with 900 mg or higher gossypol diets. Hematocrit was significantly affected at each incremental level of dietary gossypol of 600 mg/kg or higher. Serum protein did not differ for fish fed with the three lowest dietary levels of gossypol but was significantly reduced at 900 mg or higher gossypol. Macrophage chemotaxis ratio was similar for groups fed diets containing gossypol but was significantly higher than that of fish fed with the control diet. Serum lysozyme activity significantly increased at dietary gossypol levels of 900 mg or higher. Superoxide anion production as measured by nitroblue tetrazolium (NBT) reduction assay did not differ among treatments. Mortality at 14-day post-challenge with E. ictaluri significantly decreased at dietary gossypol levels of 900 mg or higher. However, antibody titer against E. ictaluri 15-day post-challenge was not affected by dietary treatments. This study indicates that juvenile channel catfish was more sensitive to gossypol toxicity than previously reported. The toxic concentration ranged from 300 to 1200 mg gossypol/kg diet depending on the parameter evaluated. However, improved macrophage chemotaxis ratio, serum lysozyme activity and resistance of catfish to E. ictaluri challenge were observed at dietary levels of 900 mg gossypol/kg or higher.

Rapid detection of columnaris disease in channel catfish (Ictalurus punctatus) with a new species-specific 16-S rRNA gene-based PCR primer for Flavobacterium columnare

A 16-S rRNA gene from the chromosomal DNA of the fish-pathogenic bacterium Flavobacterium columnare (formerly Flexibacter columnaris), strain ARS-I, was cloned, sequenced and used to design a polymerase chain reaction (PCR) primer set. The primer set amplified a specific 1193-bp DNA fragment from F. columnare strains but not from related bacteria, F. psychrophilum, F. aquatile, F. branchiophilum, or other bacterial pathogens of fish, Flexibacter maritimus, Cytophaga johnsonae, Edwardsiella ictaluri, E. tarda, Aeromonas hydrophila, and Streptococcus iniae or from the non-fish pathogen Escherichia coli. The PCR reaction conditions were optimized to permit detection of the organism from agar plates, broth culture, frozen samples, dead fish tissue, and live fish in less than 5 h (8 h, if the more sensitive nested PCR is used). DNA was extracted by a boiled-extraction method or by commercial column purification. The PCR product was detected at DNA concentrations below 0.1 ng and from as few as 100 bacterial cells. Nested PCR using universal eubacterial primers increased the sensitivity five-fold, allowing detection of F. columnare strains at DNA concentrations below 0.05 ng and from as few as 10 bacterial cells in apparently healthy, asymptomatic fish. The efficiency of this primer set was compared to the 16-S rRNA gene primer sets of Toyama et al. [Fish Pathol. 29 (1994) 271.] and that of Bader and Shotts [J. Aquat. Anim. Health 10 (1998) 311.]. The new primer set is as good or better than the previously published primer sets for detecting F. columnare in all samples and under all conditions tested.